U.S. patent application number 16/499391 was filed with the patent office on 2020-02-13 for method for electrochemical detection of mycobacteria.
The applicant listed for this patent is INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE - INRA, UNIVERSITE DE BOURGOGNE. Invention is credited to Elodie BARBIER, Alain HARTMANN, Murielle ROCHELET.
Application Number | 20200048678 16/499391 |
Document ID | / |
Family ID | 59745981 |
Filed Date | 2020-02-13 |
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United States Patent
Application |
20200048678 |
Kind Code |
A1 |
ROCHELET; Murielle ; et
al. |
February 13, 2020 |
METHOD FOR ELECTROCHEMICAL DETECTION OF MYCOBACTERIA
Abstract
The present invention relates to a novel process for biological
detection of mycobacteria via electrochemical analysis methods of
the catalytic activity of antigen 85.
Inventors: |
ROCHELET; Murielle; (DIJON,
FR) ; BARBIER; Elodie; (ROUVRES-SOUS-MEILLY, FR)
; HARTMANN; Alain; (DIJON, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITE DE BOURGOGNE
INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE - INRA |
DIJON
PARIS |
|
FR
FR |
|
|
Family ID: |
59745981 |
Appl. No.: |
16/499391 |
Filed: |
March 29, 2018 |
PCT Filed: |
March 29, 2018 |
PCT NO: |
PCT/FR2018/050769 |
371 Date: |
September 30, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 27/3275 20130101;
C12Q 1/005 20130101; C12Q 1/48 20130101; C12Q 1/04 20130101 |
International
Class: |
C12Q 1/04 20060101
C12Q001/04; C12Q 1/48 20060101 C12Q001/48; G01N 27/327 20060101
G01N027/327 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2017 |
FR |
1752787 |
Claims
1) A process for electrochemical detection of mycobacteria in a
biological sample, said process comprising the steps of: a)
selecting a substrate of at least one acyltransferase and its
cofactor; b) bringing said biological sample into contact with said
substrate and cofactor; c) electrochemically detecting the product
resulting from the catalytic activity of said at least one
acyltransferase.
2) The process as claimed in claim 1, wherein said at least one
acyltransferase is Antigen 85.
3) The process as claimed in claim 1, wherein the biological sample
is chosen from bacterial cultures, biological specimens of human or
animal origin, and environmental samples.
4) The process as claimed in claim 1, wherein said substrate of
said at least one acyltransferase is chosen from
p-aminophenyl-6-O-octanoyl-.beta.-D-glucopyranoside, and a
substrate with acyl groups having alkyl chains ranging from
C.sub.7H.sub.15 to C.sub.29H.sub.59.
5) The process as claimed in claim 1, wherein said cofactor is a
sugar chosen from the group consisting of trehalose and
D-glucose.
6) The process as claimed in claim 1, wherein the electrochemical
detection step c) is carried out by means of an amperometric
sensor.
7) The process as claimed in claim 1, wherein said biological
sample has been treated beforehand by means of a process for
isolating mycobacteria, comprising the steps of: A. placing said
biological sample in solution; B. treating with an apolar solvent
the solution obtained in step A); C. recovering the mycobacteria by
filtration or centrifugation of the solution resulting from step
B); and D. recovering the mycobacteria from the filtrate or from
the centrifugation pellet obtained at the end of step C).
8) The process as claimed in claim 7, also comprising a step of
decontaminating A') the biological sample placed in solution at the
end of step A) and before step B), and/or a step of decontaminating
C') the filtering membrane at the end of step C) and before step
D).
9) The process as claimed in claim 8, wherein step C') is carried
out with acid solutions and/or basic solutions, and/or addition of
sodium hypochlorite, and/or addition of at least one disinfecting
compound.
10) The process as claimed in claim 8, also comprising a step of
rinsing C'') the filtering membrane at the end of step C') and
before step D).
11) The process as claimed in claim 10, wherein the rinsing step
C'') is carried out with a phosphate buffer.
12) A kit for carrying out the process for electrochemical
detection of mycobacteria in a biological sample as defined in
claim 1, comprising: i. a device and the reagents for collecting
and preparing the biological sample to be tested; ii. a device
comprising a substrate of Ag85 and its cofactor for the incubation
with Ag85; iii. a device for the electrochemical detection by means
of a suitable reader.
13) The kit as claimed in claim 12, wherein the device for the
electrochemical detection of step iii) is an amperometric sensor,
preferably a screen-printed sensor.
Description
FIELD OF THE INVENTION
[0001] The subject of the present invention is a process or a
method for detecting mycobacteria, which is based on measuring
acyltransferase activity, in particular the catalytic activity of
Antigen 85, with an electrochemical analysis method.
[0002] The present invention is applicable in human and veterinary
medicine, for the diagnostic of human and animal tuberculosis and
mycobacteriosis, and also in environmental diagnosis.
[0003] In the description below, the references between square
brackets ([ ]) refer to the list of references presented at the end
of the text.
PRIOR ART
[0004] Mycobacteria belong to the phylum Actinobacteria and are
characterized by a wall rich in mycolic acids giving them
particular staining properties associated with resistance of their
wall to successive decolorings by an acid and then by 90.degree.
alcohol (AAFB: Acid-Alcohol-Fast Bacilli). Approximately 200
species belonging to the Mycobacterium genus have been identified
to date.
[0005] Among them, mycobacteria referred to as tuberculous are
necessary pathogenic bacteria with predominantly respiratory
tropism, that are responsible for tuberculosis in human beings and
animals. Non-tuberculous mycobacteria (termed atypical or
environmental) group together opportunistic bacteria responsible
for mycobacteriosis in human beings and animals. Leprosy is also a
disease caused by a Mycobacterium, Mycobacterium leprae.
[0006] Human tuberculosis is mainly due to M. tuberculosis, but can
also be caused by M. africanum, M. canettii, M. bovis in
particular. Human tuberculosis represents a major health problem
worldwide since it is the most deadly infectious disease in the
world (approximately 1.5 million deaths in 2013) with HIV (AIDS).
The WHO (World Health Organization) estimates the number of new
cases to be approximately 9 million each year.
[0007] With regard to animals, tuberculosis affects a very large
number of species: bovines, members of the goat family and also
numerous wild species, such as small rodents, for example.
[0008] Atypical mycobacteria, widely found in the environment (soil
and water), exhibit a very variable pathogenicity in human beings
and animals.
[0009] In human beings, the incidence of infections associated with
atypical mycobacteria appears to increase in industrialized
countries. They generally occur where there is a background of
local or general immunodepression causing mainly pulmonary
infections (for example M. avium and M. intracellulare, M. xenopi,
M. kansasii, M. malmoense), lymphatic infections (M. avium and
intracellulare, M. kansasii, M. scorofulaceum), skin infections (M.
marinum, M. ulcerans, M. chelonae), or even systemic infections (M.
avium and intracellulare, M. kansasii, M. haemophilum, M. xenopi,
M. gevanense), etc. In animals, some cause contagious infections
with high morbidity and mortality (paratuberculosis in bovines: M.
avium ssp. paratuberculosis and "tuberculosis" in birds: M. avium
ssp. avium for example). The persistence of atypical mycobacteria
in the environment, their resistance to detergents and the ability
of certain species to form biofilms, in particular in water
networks, may be responsible for the contamination of surface water
and water distribution networks responsible for contaminating human
beings.
[0010] The detection of mycobacteria in human beings, animals or in
the environment is based on the following techniques.
[0011] The historical detection method is based on a microscopic
examination of samples (sputum smear, ground material from lesions,
etc.) which makes it possible to demonstrate the presence of
acid-alcohol-fast bacilli (AAFBs), a partially specific
characteristic of mycobacteria. In human beings, the microscopic
examination is carried out on a biological sample smear or the
centrifugation pellet obtained after fluidization-decontamination
of contaminated pathological products. Two stainings are used:
Ziehl-Neelsen staining (conventional microscopy) and auramine
staining (fluorescence microscopy). It is a key examination since
the majority of cases of tuberculosis in countries where there is a
high incidence are diagnosed in this way in peripheral microscopy
centers. Microscopic examination also remains the starting point
for the diagnosis scheme adopted in diagnostic laboratories in
developed countries. Microscopic examination is easy to implement
(little material, personnel not highly qualified) with the result
being provided rapidly (2 to 3 hours) and at low cost. However,
this examination has several drawbacks: operator-dependent
implementation, subjective interpretation of the result, lack of
sensitivity (detection of 50% to 70% of pulmonary tuberculosis
cases), and also a lack of identification of the species involved.
The performance levels thereof are even lower in patients infected
with HIV and children (specimens with few bacteria).
[0012] With regard to the bacteriological detection of tuberculosis
and of mycobacteriosis in human beings and animals, and also that
of environmental contamination, the reference technique remains
culture on a suitable medium (solid: Coletsos, Lowenstein-Jensen,
Middlebrook 7H11 or liquid: Middlebrook 7H9) optionally
supplemented with antibiotics and antifungals. The culturing of the
bacterium starting from sputum, ground tissue matter, other
biological samples or environmental specimens is commonly used and
has the advantage of being sensitive. Automated liquid culture
systems of Bactec MGIT.TM. (Becton Dickinson) or BacT/Alert.RTM.
(BioMerieux) type combine incubation and spectroscopic measurement
of bacterial growth. However, this method allows only a delayed
diagnosis since, from a microbiological and culture point of view,
tuberculous mycobacteria and some atypical mycobacteria are
slow-growing microorganisms: at least 1 to 6 weeks of incubation at
37.degree. C. are necessary in order to observe growth of the
bacterium on the culture media. Whatever the nature of the sample,
a prior decontamination treatment optionally combined with
fluidization with physicochemical agents (N-acetylcysteine--sodium
hydroxide, sodium hypochlorite, acids, detergents) is essential
before it is cultured, in order to prevent the development of
fast-growing microorganisms, reducing the sensitivity of the
method. Following the culture, the identification of the species
can be carried out by DNA hybridization techniques optionally
coupled with PCR, gene sequencing, genotyping (insertion sequences,
spoligotyping, etc.), by identification of biomarkers (analysis of
mycolic acids by liquid-phase chromatography, protein profile by
MALDI-TOF mass spectrometry for example). However, all these
techniques require expensive laboratory equipment and highly
qualified staff and are not therefore suitable for outsourced and
rapid diagnosis of mycobacterial infections.
[0013] Because of their rapidity (result provided during the day),
molecular biology methods are also today widely used in diagnostic
laboratories both for human beings and for animals and the
environment. Based on the specific amplification of target
mycobacterial genes, they allow both the detection of the bacterium
and its identification, or even its possible resistance to
antibiotics (human diagnosis only). However, their use generally
requires suitable infrastructure, expensive equipment and also
qualified staff (DNA extraction and interpretation of the results).
The GeneXpert.RTM. technology developed by the Cepheid laboratory
for the molecular diagnosis of human tuberculosis limits the
drawbacks mentioned above by virtue of the use of an automated
device which carries out all the steps without human intervention.
The result obtained in 2 hours makes it possible to detect the
presence of a tuberculous mycobacterium and also its potential
resistance to rifampicin, a frontline antibiotic used in the
treatment of human tuberculosis. However, its price constitutes a
curb on its generalization in countries with low revenues.
Furthermore, this technology does not make it possible to carry out
more than about twenty analyses per day.
[0014] In animals, the diagnosis of tuberculosis is carried out
post-mortem after prophylactic screening or a discovery of lesions
in the abattoir. It is carried out on ground material from lesions
or from lymph nodes. As in human beings, the samples can be
cultured after decontamination and/or can be analyzed by molecular
biology methods. The limits of this detection are identical to
those mentioned above: delayed production of the result with
culture and expensive molecular biology automated devices with
qualified staff.
[0015] Regarding the detection of mycobacteria in the environment,
the search for said mycobacteria is not standardized and no
standard is currently available. Culture on a suitable medium after
chemical decontamination comes up against the same limits as that
of biological samples: slow growth with delayed production of the
result and contamination of the culture media by fast-growing
bacteria in particular. Molecular biology has made it possible to
bypass this culture step, but still does not allow suitable
quantification.
[0016] With a view to proposing new methods of identifying
mycobacteria, the detection of specific antigens, such as Ag MPT64
or those of the Antigen 85 (Ag85) complex have been envisioned. To
do this, an immunochromatographic test based on the identification
of Ag MPT64 after culture (present in tuberculous mycobacteria,
absent in atypical mycobacteria) has been sold (SD Bioline TB Ag
MPT64, Standard Diagnostic, Inc.). ELISA assays used for the
detection of Ag85 in liquid culture filtrates, serum or in
cerebrospinal fluid (Phunpae et al., Diagn. Microbiol. Infect.
Dis., 78(3): 242-248, 2014 [1]; Kashyap et al., BMC infectious
diseases, 7:74, 2007 [2]; Kashyap et al., Clin Diagn Lab Immunol.,
12(6):752-758, 2005 [3]) are described in the literature.
[0017] The Antigen 85 (Ag85) complex is composed of three secreted
homologous proteins: Antigen 85A (Ag85A), Antigen 85B (Ag85B), and
Antigen 85C (85C) which share a high sequence identity (68-79%) in
their secreted mature forms. They are mycolyltransferases (enzymes
having a molecular weight of approximately 30 000 Da) which are
involved specifically in the construction and maintenance of the
walls of Corynebacteriales--order to which the Mycobacterium genus
belongs--by catalyzing the transfer of mycolic acid onto
polysaccharide structures (arabinogalactan, trehalose). More
generally, Ag85 is an acyltransferase which is not only capable of
transferring mycolyl groups, but also other acyl groups.
[0018] The detection and the activity of Ag85 being widely studied
for the search for and evaluation of new methods of diagnosis and
of monitoring of the efficacy of antitubercular chemotherapy
treatments (Elamin et al., J Microbiol Methods, 79(3):672-678, 2002
[4]) several spectrophotometric methods (UV-visible, fluorescence,
etc.) have been described for the assaying of this protein via the
measurement of its acyltransferase activity (Boucau et al.,
Analytical Biochem., 385: 120-127, 2009 [5]; Favrot et al., J.
Biol. Chem., 289(36): 25031-25040, 2014 [6]).
[0019] International application WO 2011/030160 [7] describes a
method for detecting the presence of mycobacteria in an organism or
a biological sample via the demonstration of the catalytic activity
of Ag85 during the culture step. To do this, molecular probes
consisting of a labelled polysaccharide (trehalose and other
saccharide derivatives) (radiotracer, fluorophore, nanoparticles,
biotin) have been synthesized and added to the culture medium in
order to be incorporated into the bacterial wall during bacterial
growth by virtue of the transferase activity of Ag85. At the end of
this step, the bacteria are rinsed and isolated from the culture
medium and then detected using a suitable technique (scintillation
counter, fluorimeter, microscopy, NMR, in vivo imaging techniques,
etc.). However, this method, which allows the detection of viable
mycobacteria by labeling them, can only be envisaged for the
analysis of very contaminated samples (about 10.sup.7
bacilliml.sup.-1) or after quite a long culture step. Furthermore,
several steps of rinsing the bacteria are obligatory in order to
remove the excess labeled probe not incorporated. Finally, the
detection of the marker is carried out using delicate and expensive
laboratory instrumental techniques which require qualified staff to
implement them and to interpret the results.
[0020] Patent application CN102087283 [8] describes a method of
electrochemical detection of M. tuberculosis using an enzymatic
immunosensor based on a solution of chitosan, gold nanoparticles
and an antibody specific for the M. tuberculosis cell wall. The
quantitative measurement is carried out by comparing the signal of
the product generated by alkaline phosphatase in the presence of
.alpha.-naphthyl phosphate before and after incubation with the
sample. However, although this method has been applied to the
detection of M. tuberculosis in milk samples, its use in routine
diagnosis cannot be envisioned. This is because the use of vitreous
carbon electrodes for the construction of the immunosensor is very
restrictive: polishing of the surface and cleaning in a piranha
mixture (sulfuric acid and hydrogen peroxide) before each new use.
Furthermore, the preparation of the sensitive surface of the
immunosensor requires several steps: 1) electrodeposition of a
solution containing gold nanoparticles, chitosan and a goat
anti-mouse antibody labeled with an alkaline phosphatase, then 2)
incubation of a solution of anti-M. tuberculosis antibody produced
in mice. Finally, once constructed, the immunosensor must be stored
at 4.degree. C.
[0021] There is thus a need for a method for detecting mycobacteria
that is simple and rapid to carry out and that overcomes the
drawbacks of the processes of the prior art.
DESCRIPTION OF THE INVENTION
[0022] In order to meet this need for a more effective diagnostic
test for human, animal and/or environmental tuberculosis for which
at the current time there is no suitable solution, the inventors
have developed a new electrochemical method capable of rapidly
detecting (obtaining the results in approximately 2 to 5 h) the
presence or absence of mycobacteria in samples such as, for
example, in a culture medium and in human respiratory specimens:
the EDMYC (Electrochemical Detection of MYCobacteria) method.
[0023] Thus, the inventors have developed a new method for
electrochemical detection of mycobacteria via the electrochemical
measurement of the acyltransferase activity in the mycobacteria, in
particular of the catalytic activity of Ag85 in the presence of a
substrate of the enzyme, e.g.
p-aminophenyl-6-O-octanoyl-.beta.-D-glucopyranoside (p-AP-OG), and
of a cofactor or co-substrate, e.g. trehalose. Indeed, since Ag85
is very widely excreted by mycobacteria, e.g. by Mycobacterium
tuberculosis and Mycobacterium bovis, in liquid culture media, the
detection of the acyltransferase activity, in particular of the
catalytic activity of Ag85, in the culture medium makes it possible
to demonstrate the presence of mycobacteria.
[0024] The principle of the invention is based on the fact that
acyltransferases such as Ag85 hydrolyze the ester bond of the
substrate, and transfer the acyl group thus released onto the
cofactor. The product is then detected by voltammetry. According to
one particular embodiment, the present invention is based on the
capacity of acyltransferases, in particular of Ag85, to hydrolyze
the ester bond of p-AP-OG and to transfer the octanoyl group of
p-AP-OG onto a sugar, e.g. trehalose, according to a ping-pong
mechanism, in order, respectively, to generate
p-aminophenyl-.beta.-D-glucopyranoside (p-AP-G) and to form
acyltrehalose (FIG. 1). The difference between the potentials of
the oxidation peaks of p-AP-OG and of p-AP-G that is observed on
the voltammograms (FIG. 2), which is explained respectively by the
presence or the absence of the octanoyl group on the molecule, thus
makes possible the specific detection of the acyltransferase
activity, in particular of the catalytic activity of Ag85, in the
presence of p-AP-OG. The intensity of the p-AP-G oxidation peak,
chosen as analytic response, is proportional to the amount of
acyltransferases, in particular of Ag85, and thus to that of the
mycobacteria present in the sample analyzed.
[0025] Thus, the inventors have developed a simple and rapid method
for detecting mycobacteria and their viability with or without a
prior culture step.
[0026] To date, the proof of concept of the method has been
successfully demonstrated with the detection of several
mycobacterial species frequently encountered in pulmonary
infections, including M. tuberculosis--the principal agent of
tuberculosis in human beings--in liquid and solid cultures. The
method has numerous advantages compared with microscopic
examination, such as the simplicity of its implementation or else
an easy interpretation of the results (numerical measurement) with
a small, portable and inexpensive piece of equipment. In addition,
compared with optical methods, the electrochemical technique proves
to be particularly advantageous since it allows the analysis of
cloudy or colored samples, with the possibility of offering a
quantified measurement, with good sensitivity, by means of
single-use screen-printed sensors. Thus, it perfectly satisfies the
specifications imposed by the WHO for a test capable for example of
replacing the microscopic examination of sputum smears.
[0027] In addition, the inventors have demonstrated an improvement
in the specificity of the detection method with respect to
mycobacteria and Ag85 by proposing 1) the use of a substrate of the
enzyme with acyl groups having carbon chains longer than that of
p-AP-OG, for example alkyl chains ranging from C.sub.7H.sub.15 to
C.sub.29H.sub.59, and/or 2) a method for extracting and
decontaminating actual samples in order to isolate the
mycobacteria.
[0028] A subject of the present invention is thus a process for
electrochemical detection of mycobacteria in a biological sample,
said process comprising the steps of: [0029] a) selecting a
substrate of at least one acyltransferase and its cofactor; [0030]
b) bringing said biological sample into contact with said substrate
and cofactor; [0031] c) electrochemically detecting the product
resulting from the catalytic activity of said at least one
acyltransferase.
[0032] According to one particular embodiment of the detection
process of the present invention, the biological sample is chosen
from the group consisting of: bacterial cultures, biological
specimens of human or animal origin, environmental samples, etc. A
bacterial culture may for example be obtained on a nutritive agar
or in a liquid culture medium, according to techniques well known
to those skilled in the art. A biological specimen of human origin
may for example be a sample of pulmonary origin (sputum, bronchial
secretions, biopsy), a blood sample, a cerebrospinal fluid sample,
a urine sample, a sample of intestinal origin (intestinal biopsy,
feces), and also any other tissue sample. A biological specimen of
animal origin may for example be a tissue sample (lymph node, lung,
liver, spleen, etc.), a sample of feces or a milk sample. A sample
of environmental origin may for example be a sample of waste water,
or of hospital waste water, a sample of treated waste water, a
sample of sludge resulting from the treatment of waste water or a
soil sample.
[0033] According to one particular embodiment of the detection
process of the present invention, the acyltransferase substrate is
chosen from the group consisting of:
p-aminophenyl-6-O-octanoyl-.beta.-D-glucopyranoside, and substrates
with acyl groups having alkyl chains ranging from C.sub.7H.sub.15
to C.sub.29H.sub.59.
[0034] According to one particular embodiment of the detection
process of the present invention, the cofactor is a sugar chosen
from the group consisting of: trehalose, D-glucose.
[0035] Preferably, the substrate is
p-aminophenyl-6-O-octanoyl-.beta.-D-glucopyranoside, and the
cofactor is trehalose. The product formed after enzymatic
hydrolysis by the acyltransferases, in particular by Ag85, is
p-aminophenyl-.beta.-D-glucopyranoside.
[0036] According to one particular embodiment of the detection
process of the present invention, the electrochemical detection
step c) is carried out by means of an amperometric sensor, which is
optionally chemically modified (e.g. with carbon nanotubes,
graphene). Preferably, said sensor is a screen-printed sensor,
which is preferentially single-use.
[0037] In accordance with the invention, an electrochemical
analysis means for carrying out the invention can be a
potentiometric measurement, an impedance measurement, a coulometric
measurement or an amperometric measurement.
[0038] According to one advantageous embodiment of the detection
process of the present invention, the electrochemical analysis is
carried out by an amperometric measurement.
[0039] For the purposes of the present invention, the term
"amperometric measurement" is intended to mean a measurement of the
electric current as a function of a potential difference
established between the working electrode and the reference
electrode.
[0040] The measurement of the electric current can be carried out
by means of known amperometric techniques, preferentially by
potential sweep voltammetry which may be linear, cyclic, or pulse
voltammetry or else of the potential step type, such as
chronoamperometry.
[0041] In one particularly advantageous embodiment of the detection
process of the present invention, the presence of
p-aminophenyl-.beta.-D-glucopyranoside is measured by cyclic or
linear voltammetry.
[0042] The use of these techniques requires an assembly which may
be a two-electrode or even three-electrode assembly, that is to say
an assembly comprising a working electrode, a reference electrode
and optionally an auxiliary electrode (counter electrode). The
working electrode, the surface of which serves as a site for
electron transfer, can be based on carbon or based on a noble metal
or else based on metal oxide. The reference electrode is an
electrode of which the potential is constant, which makes it
possible to impose a precisely defined potential on the working
electrode. The reference electrode may be an Ag/AgCl electrode. The
counter electrode, which makes it possible to establish the passage
of the electric current with the working electrode, can be
fabricated with an inert material, such as platinum or carbon.
Those skilled in the art will know how to choose and combine the
appropriate electrodes according to their general knowledge.
[0043] With regard to the method of manufacturing the electrodes,
the screen-printing technique is preferable, although other methods
of industrial fabrication, such as rotagravure, inkjet printing, 3D
printing or optionally photolithography, can be envisioned.
Electrodes obtained by screenprinting are particularly well suited
because they can be produced in bulk at low cost, and thus can
optionally be single-use. Furthermore, their geometric shape and
also their size can be easily modulated. These electrodes can be
screenprinted in the form of a sensor and optionally integrated
into the bottom of the wells of a microplate or of other supports
or systems allowing the filtration of the bacterial suspensions and
the incubation of
p-aminophenyl-6-O-octanoyl-.beta.-D-glucopyranoside and of
trehalose.
[0044] According to one particular embodiment of the detection
process of the present invention, the amperometric measurement is
carried out with a screen-printed sensor. It makes it possible to
perform the measurement in a small volume of solution of about a
few microliters.
[0045] According to one particular embodiment of the detection
process of the present invention, the amperometric measurement is
carried out with a device involved three electrodes: an Ag/AgCl
reference electrode, a carbon working electrode and a carbon
counter electrode.
[0046] According to another particular embodiment of the detection
process of the present invention, the amperometric measurement is
carried out with a screen-printed sensor comprising an Ag/AgCl
reference electrode, a carbon working electrode and a carbon
counter electrode.
[0047] The presence of p-aminophenyl-.beta.-D-glucopyranoside is
indicated by the presence of an anodic oxidation current in an
interval of potentials and the absence of said current for a
control devoid of hydrolyzed
p-aminophenyl-6-O-octanoyl-.beta.-D-glucopyranoside.
[0048] When the p-aminophenyl-.beta.-D-glucopyranoside is subjected
to a measurement by cyclic voltammetry, its presence is indicated
by an anodic oxidation current peak specific to
p-aminophenyl-.beta.-D-glucopyranoside in a determined interval of
potentials (+0.3 to +0.5 V vs. Ag/AgCl).
[0049] Preferably, the biological sample to be tested is prepared
so as to isolate the mycobacteria that it contains while at the
same time eliminating a maximum amount of contaminants before the
contacting and detection steps. To do this, an extraction step with
an apolar solvent such as, for example, hexane is necessary in
order to selectively isolate the mycobacteria--the wall of which is
very hydrophobic--from the sample previously placed in solution in
a fluidizing agent such as N-acetylcysteine for a respiratory
specimen or in a phosphate buffer with a neutral pH for a soil
sample, for example. In the case of complex samples such as soil,
where millions of different bacterial species can coexist, the
extraction step can be followed by a decontamination of the extract
with, for example, an acid (HCl, H.sub.2SO.sub.4) and/or a base
(NaOH) or a quaternary ammonium.
[0050] Thus, a subject of the present invention is also a process
for isolating mycobacteria from a biological sample, said process
comprising the steps of: [0051] a) placing said biological sample
in solution; [0052] b) treating with an apolar solvent the solution
obtained in step a); and [0053] c) recovering the mycobacteria by
filtration or centrifugation of the solution resulting from step
b); and [0054] d) recovering the mycobacteria from the filtrate or
from the centrifugation pellet obtained at the end of step c).
[0055] According to one particular embodiment of the process for
isolating mycobacteria of the present invention, the extraction
step b) is carried out with a solution of hexane or a
hexane-isopropanol mixture.
[0056] According to one particular embodiment of the process for
isolating mycobacteria of the present invention, the process can
also comprise a step of decontaminating a') the biological sample
placed in solution at the end of step a) and before step b), and/or
a step of decontaminating c') the filtering membrane at the end of
step c) and before step d), with acidic solutions (e.g. solution of
hydrochloric acid) and/or basic solutions (e.g. solution of sodium
hydroxide or of quaternary ammonium), and/or addition of sodium
hypochlorite, and/or with at least one other disinfecting compound
(e.g. chlorhexidine or squalamine).
[0057] According to one particular embodiment of the process for
isolating mycobacteria of the present invention, step c') can be
followed, before step d), by a rinsing step c''), for example in
the presence of phosphate buffer, in order to remove the sodium
hypochlorite (bleach) from the filter (preferably made of
Teflon).
[0058] According to one particular embodiment of the process for
isolating mycobacteria of the present invention, step d) of
recovering the mycobacteria is carried out by scraping the filter
with a loop (wire loop) in order to detach the cells from the
filter. The mycobacteria thus recovered are then cultured in a
suitable medium (enriched and supplemented medium 7H11), for
approximately two months, at 37.degree. C., in order to allow
counting thereof.
[0059] A subject of the present invention is also a kit for
carrying out the process for electrochemical detection of
mycobacteria in a biological sample according to the present
invention, said kit comprising: [0060] a) a device and the reagents
for collecting and preparing the biological sample to be tested;
[0061] b) a device comprising a substrate of Ag85 and its cofactor
for the incubation with Ag85; [0062] c) a device for the
electrochemical detection by means of a suitable reader.
[0063] For the purposes of the present invention, the term "device"
of step a) is intended to mean a sealed container, which is
preferably single-use, for example a single-use tube or column
equipped with a filtration system in which the steps of diluting
the sample, extracting, decontaminating if necessary and recovering
the mycobacteria are carried out.
[0064] For the purposes of the present invention, the term "device"
of step b) is intended to mean a sealed container, which is
preferably single-use, for example a single-use tube equipped with
a filtration system in which the incubation of the mycobacteria
with the substrate and the co-substrate is carried out.
[0065] For the purposes of the present invention, the expression
"device for the electrochemical detection" of step c) is intended
to mean for example an amperometric sensor, which is preferably
screenprinted and single-use, for example those sold by the
companies Dropsens and Palmsens. The amperometric sensor can
optionally be integrated into the device of step b.
[0066] By way of example of a suitable reader, mention may be made
of portable readers based on the principle of the blood glucose
reader, for example those sold by the companies Dropsens and
Palmsens and which make it possible to carry out the measurements
in a few seconds.
BRIEF DESCRIPTION OF THE FIGURES
[0067] FIG. 1 represents the scheme of the principle of the
enzymatic reaction catalyzed by Ag85 with p-AP-OG and trehalose as
substrate and substrate, respectively.
[0068] FIG. 2 represents the linear voltammograms (v=50 mVs.sup.-1)
of a solution of substrate (p-AP-OG; S) and of product (p-AP-G;
dashed curve; P) at 5.times.10.sup.-4 M in PBS (pH 7.5)--0.2%
DMSO.
[0069] FIG. 3 represent the linear voltammograms (v=50 mVs.sup.-1)
recorded for a solution of Ag85 (16 .rho.gml.sup.-1) incubated for
4 h at 37.degree. C. with p-AP-OG (2.times.10.sup.-4 M) and
trehalose (10 mM) in PBS in the presence (dotted curve) and in the
presence (curve as a continuous line) of p-AP-G (10.sup.-4 M). The
curve as a line --- corresponds to the voltammogram obtained after
incubation under the same conditions of a solution of p-AP-OG
(2.times.10.sup.-4 M) in PBS. Reaction volumes=15 .mu.l.
[0070] FIG. 4 represent the steps carried out during the detection
of Ag85 in the supernatant of a culture of mycobacteria.
[0071] FIG. 5 represents the linear voltammograms (v=50 mVs.sup.-1)
recorded after carrying out the protocol of FIG. 4 for the analysis
of 5 ml of 7H9 medium (curve as a continuous line) and 5 ml of M.
bovis BCG culture (1.1.times.10.sup.7 cfuml.sup.-1).
[0072] FIG. 6 represents the steps carried out during the detection
of Ag85 in the bacterial pellet resulting from a culture of
mycobacteria.
[0073] FIG. 7 represents the linear voltammograms (v=50 mVs.sup.-1)
recorded by analyzing 10 ml volumes of a liquid culture of M. bovis
BCG at 2.times.10.sup.6 cfuml.sup.-1 according to the protocols of
FIGS. 4 and 6. The curve as a line --- corresponds to the response
recorded for the supernatant and that as a continuous line was
obtained for the analysis of the bacterial pellets.
[0074] FIG. 8 represents the linear voltammograms (v=50 mVs.sup.-1)
recorded by analyzing 1 ml of 7H9 culture medium+OADC (negative
control; curve as a continuous line) containing (A) M.
intracellulare, (B) M. avium and (C) M. xenopi at .about.10.sup.6
bacilliml.sup.-1 (curved as a line ---) analyzed according to the
protocol described in section 1.5.
[0075] FIG. 9 represents the linear voltammograms (v=50 mVs.sup.-1)
recorded for the qualitative analysis of a liquid culture of M.
tuberculosis (A) culture supernatant, (B) bacterial pellet, and
that (C) of isolated colonies of M. tuberculosis according to the
protocol described in section 1.6. The curves as a line ---
correspond to the M. tuberculosis response while the curves as a
continuous line were obtained for the negative control.
[0076] FIG. 10 represents the linear voltammograms (v=50
mVs.sup.-1) recorded after carrying out the protocol described in
section 1.7 for the analysis of 1 ml of sputum inoculated with
10.sup.6 M. intracellulare bacilli (dashed curve) or not incubated
(curve as continuous line).
[0077] FIG. 11 represents the linear voltammogram (v=50 mVs.sup.-1)
recorded by analyzing 1 ml of M. intracellulare culture at 10.sup.5
bacilliml-according to the protocol described in section 1.5. and
also the values i p-AP-OG and i p-AP-G required for calculating the
analytical response R.
EXAMPLES
Example 1: Materials and Methods
[0078] 1.1. Reagents and solutions [0079] the
p-aminophenyl-6-O-octanoyl-.beta.-D-glucopyranoside
(C.sub.20H.sub.31NO.sub.7; p-AP-OG) and also the
p-aminophenyl-6-.beta.-D-glucopyranoside (C.sub.12H.sub.17NO.sub.6;
p-AP-G) were synthesized at the Institut de Chimie Moleculaire
(Molecular Chemistry Institute) of the Universite de Bourgogne.
[0080] the trehalose and the dimethyl sulfoxide (DMSO) were
supplied by Sigma-Aldrich. [0081] the Mycobacterium tuberculosis
Ag85B (Ag 85; ab73632) was purchased from Abcam and reconstituted
according to the supplier's recommendations. [0082] the hexane and
the isopropanol come from the Carl Roth laboratory. [0083] the
phosphate buffer (16.7 mM NaH.sub.2PO.sub.4.2H.sub.2O; 33.3 mM
Na.sub.2HPO.sub.4.12H.sub.2O [0084] pH 7.5, 50 mM) was prepared
with Milli-Q 18 MO water (Millipore System). [0085] the liquid
medium (Middlebrook 7H9 Broth Base, Fluka) and solid medium
(Middlebrook 7H11 Agar Base, Fluka) and also the constituents of
the enrichment product (oleic acid, bovine albumin, dextrose and
catalase) were supplied by Sigma-Aldrich. The tryptone (casein
peptone) was supplied by VWR. The heat-inactivated bovine serum was
supplied by Dutscher. [0086] the BD BACTEC.TM. MGIT.TM. liquid
medium tubes (mycobacterial growth indicator tubes) were supplied
by Becton Dickinson, as were the BACTEC.TM. MGIT.TM. growth
supplement and the lyophilized BBL MGIT.TM. PANTA antibiotic
complex. The medium was prepared according to the distributor's
recommendations.
[0087] 1.2. Strains and Culture Media [0088] the strains of
Mycobacterium intracellulare, M. avium ssp. avium, M. bovis BCG
strain Pasteur (avirulent vaccine strain) and M. xenopi were
supplied by the Laboratoire National de Reference [French National
Reference Laboratory] for bovine tuberculosis of Maisons-Alfort.
The strain M. avium ssp. paratuberculosis K10 was supplied by the
INRA [French National Institute for Agronomic Research] of Tours.
These strains were handled in an L2 containment laboratory. [0089]
the strain of M. tuberculosis H37Rv originates from the Laboratory
associated with the Centre National de Reference des Mycobacteries
et de la Resistance des Mycobacteries aux Antituberculeux [French
National Reference Center for mycobacteria and the resistance of
mycobacteria to anti-tuberculosis agents] (Hopitaux Universitaires
[University hospitals] St Louis--Lariboisiere--F. Widal). Since the
M. tuberculosis strains belong to "Class 3" infectious risks
category, all the experiments with the strain M. tuberculosis H37Rv
were carried out in an L3 laboratory. [0090] the strains of
Staphylococcus aureus (DMSZ20231), Staphylococcus epidermidis
(DSMZ20044), Pseudomonas monteilii (DMSZ14164), Enterococcus
faecalis (DMSZ20478), Enterococcus faecium (DMSZ20477), Escherichia
coli (DMSZ30083). and Stenotrophomonas maltophilia (DMSZ50170) were
ordered from the Leibniz Institute DSMZ-German Collection of
Microorganisms and Cell Cultures. [0091] the strains of
Streptococcus pyogenes, Pseudomonas aeruginosa, Pseudomonas
fluorescens, Rhodococcus corallinus, Achromobacter xylosoxidans,
Citrobacter freundii, Enterobacter cloacae and Klebsiella
pneumoniae were kindly supplied by the INRA and the CHU [University
hospital center] of Dijon. [0092] the liquid culture medium was
prepared by diluting 5.9 g of Middlebrook 7H9 Broth Base and 1.25 g
of tryptone in 1 l of milli-Q water, then autoclaved for 15 minutes
at 121 degrees. [0093] the solid culture medium was prepared by
diluting 18.9 g of Middlebrook 7H11 Agar Base in 800 ml of milli-Q
water, then autoclaved for 15 minutes at 121 degrees.
[0094] With the aim of promoting the growth of the mycobacteria,
the liquid and solid culture media were enriched with 10% of a
mixture consisting of oleic acid, albumin, dextrose and catalase
(OADC; Table 1). The 7H11 medium was also supplemented with 10% of
heat-inactivated bovine serum.
[0095] Oleic acid and long-chain fatty acids are essential for
mycobacteria metabolism. Dextrose is an energy source. Catalase
allows neutralization of peroxides, which can be toxic to bacteria.
Albumin plays a protective role against toxic agents.
TABLE-US-00001 TABLE 1 Composition of the OADC enrichment
Components Dextrose 20.0 g Bovine albumin 50.0 g Oleic acid* 0.6
Catalase* 0.003 g Water 1 l
[0096] 1.3. Sensors and Measurement Apparatus
[0097] The electrochemical measurements were carried out by linear
voltammetry (v=50 mVs.sup.-1) with a 910 PSTAT mini potentiostat
(Metrohm, France) powered through the USB connection of the
computer and controlled by the PSTAT software (version 1.0). To do
this, drops of solution of 30-50 .mu.l were deposited on the
surface of single-use screen-printed carbon sensors supplied by
Dropsens (DRP-110) and connected to the potentiostat via the
connector (DRP-DSC). The amperometric detection of the product
generated during the reaction catalyzed by Ag85 was always carried
out after a step of filtration of the reaction mixture with a
filtration device (Microcon 10 kDa, Millipore). All the potentials
are measured relative to the Ag/AgCl reference electrode.
[0098] 1.4. Detection of Ag85 in the Supernatant of a Liquid
Culture of M. bovis BCG
[0099] The principle of this 7-step protocol is shown schematically
in FIG. 4. 4 ml of three-week-old culture of M. bovis BCG were
centrifuged at 4 000.times.g for 6 min (step 1). The supernatant
was recovered (step 2) and deposited in the reservoir of a
filtration device (Amicon.RTM. Ultra 4 ml (porosity of 50 kD
allowing the Ag85 Complex to pass through, Merck Millipore,
France)) in order to remove the interfering proteins from the
culture medium, in particular the BSA and catalase, and centrifuged
for 10 min at 7 000.times.g (step 3). The filtrate is deposited in
the reservoir of a second Amicon.RTM. Ultra 4 ml with a porosity of
10 kD, allowing the concentration of the Ag85 Complex after
centrifugation for 10 min at 7 000.times.g. The filtering membrane
is rinsed with 500 .mu.l of PBS and centrifuged according to the
same parameters as previously (step 4). The volume retained by the
membrane (200-250 .mu.l) is then deposited in a Microcon.RTM.
device with a porosity of 10 kD, centrifuged for 20 min at 14
000.times.g and then rinsed with PBS (step 5). The filtration
device is turned upside down and centrifuged for 3 min at 1
000.times.g in order to detach the Ag85 Complex (final volume of
approximately 80 .mu.l) (step 6). 10 .mu.l of a solution of
trehalose at 5.times.10.sup.-3 M in PBS and 10 .mu.l of a solution
of p-AP-OG at 2.times.10.sup.-3M in PBS are finally added to the
filtrate. After incubation at 37.degree. C. for 24 h, the reaction
medium is filtered on a Microcon.RTM. device with a porosity of 10
kD (15 min. at 14 000.times.g) and then 30 .mu.l of filtrate are
deposited on the sensor and analyzed by voltammetry (step 7).
[0100] 1.5. Detection of Ag85 in the Bacterial Pellet Obtained from
the Liquid Cultures of M. intracellulare, M. Avium and M.
Xenopi
[0101] Volumes of 1 ml of liquid culture of each mycobacterial
species (.about.10.sup.6 bacilliml.sup.-1) were centrifuged at 6
000.times.g for 6 minutes. Once the supernatant had been removed,
the bacterial pellets were rinsed with 1 ml of PBS and then
centrifuged at 6 000.times.g for 6 minutes. After removal of the
supernatant, 10 .mu.l of p-AP-OG at 2.times.10.sup.-3 M, and 10
.mu.l of trehalose at 5.times.10.sup.-3 M are added to the
bacterial pellets. After a step of incubation for 4 hours at
37.degree. C. with shaking, the electrochemical measurement of the
product of the enzymatic reaction was carried out according to the
protocol described in section 1.3.
[0102] A negative control (enriched 7H9 medium without bacteria)
was analyzed in duplicate in the same way.
[0103] 1.6. Detection of Ag85 in M. tuberculosis
[0104] 1.6.1. The Liquid Culture
[0105] Two samples of 7.5 ml of liquid culture of M. tuberculosis
(.about.10.sup.7 bacilliml.sup.-1) were centrifuged at 7
000.times.g for 10 minutes. In order to separately analyze the
bacterial pellet and the supernatant, the latter was transferred
into sterile tubes.
[0106] Analysis of the Bacterial Pellet:
[0107] Each bacterial pellet was rinsed by adding 100 .mu.l of PBS,
then the liquid was removed by turning the tube upside down on an
absorbent paper (Whatman). 20 .mu.l of p-AP-OG at 2.times.10.sup.-3
M and 20 .mu.l of trehalose at 5.times.10.sup.-3M were added to the
tubes and incubated for 4 hours at 37.degree. C. without shaking.
Negative controls (PBS) were analyzed in duplicate in the same way.
The electrochemical measurement of the product of the enzymatic
reaction was carried out according to the protocol described in
section 1.3.
[0108] Analysis of the Supernatant:
[0109] Six ml of each culture supernatant were centrifuged at 7
000.times.g for 20 min in an Amicon 50 kDa filtration device
(Millipore). The filtered liquid was transferred into an Amicon 10
kDa filtration device and then centrifuged at 7 000.times.g for 20
min. The filter was then rinsed with 1 ml of PBS and then
centrifuged at 7 000.times.g for 20 min. The volume of residual
liquid remaining on the filter was transferred into a tube to which
30 .mu.l of p-AP-OG at 2.times.10.sup.-3 M and 30 .mu.l of
trehalose at 5.times.10.sup.-3 M were added. The reaction mixture
was incubated for 4 hours at 37.degree. C. without shaking. A
negative control (enriched 7H9 medium without bacteria) was
analyzed in duplicate in the same way. The electrochemical
measurement of the product of the enzymatic reaction was carried
out according to the protocol described in section 1.3.
[0110] 1.6.2. The Isolated Colonies
[0111] A few colonies taken from a 7H11 agar were deposited in a 2
ml tube. The colonies were rinsed with PBS, the tubes were
centrifuged and the supernatant was removed. 20 .mu.l of p-AP-OG at
2.times.10.sup.-3 M and 20 .mu.l of trehalose at 5.times.10.sup.-3
M were added to the tubes and incubated for 4 hours at 37.degree.
C. without shaking. A negative control (PBS) was analyzed in
duplicate in the same way. The electrochemical measurement of the
product of the enzymatic reaction was carried out according to the
protocol described in section 1.3.
[0112] 1.7. Extraction and Detection of M. intracellulare in a
Sample of Respiratory Origin
[0113] Respiratory specimens (sputum, tracheal aspiration and
bronchial aspiration products) from nontuberculous patients were
supplied by the CHU [University hospital center] of Dijon. The
samples were fluidized beforehand by the CHU of Dijon with the
Digest-EUR kit (Eurobio).
[0114] Volumes of 1 ml of respiratory sample were dispensed into
sterile 15 ml tubes and then incubated with 200 .mu.l of a liquid
culture of M. intracellulare containing 10.sup.6 bacilli or 200
.mu.l of sterile liquid medium (negative control) overnight at
37.degree. C.
[0115] Extraction of M. intracellulare
[0116] 9 ml of hexane-isopropanol mixture (3:2, v/v) were added to
each respiratory specimen tube and stirred for 1 minute. After a
centrifugation step at 3 000.times.g for 2 minutes, the supernatant
(that is to say the hexane, and the interface) was removed and then
vacuum-filtered on a membrane (Durapore, 25 mm; 0.45 .mu.M). Once
rinsed with PBS, the membrane was placed in a small polyethylene
bag with welded zip closure having the dimensions of the
membrane.
[0117] Electrochemical Detection of M. intracellulare in the
Extract
[0118] A volume of 100 .mu.l of the mixture of substrate at
2.times.10.sup.-3 M and trehalose at 5.times.10.sup.-3 M, prepared
in PBS, was introduced into the bag before it was closed. After an
incubation step at 37.degree. C. for 4 hours, the electrochemical
measurement of the product of the enzymatic reaction was carried
out according to the protocol described in section 1.3.
[0119] 1.8. Extraction and Detection of M. bovis BCG in a Soil
Sample
[0120] Extraction of M. bovis BCG
[0121] The soil microcosms were prepared from a clay-loam soil with
pH 7.75, autoclaved twice for 15 min at 121.degree. C. with an
interval of 48 h, in order to get rid of the endogenous microflora.
Microcosms of 5 g of sterile soil were prepared in 45 ml Falcon
tubes, then inoculated with M. bovis BCG (2.6.times.10.sup.6 cfu).
After incubation for 12 h at ambient temperature in the dark, each
microcosm was subjected to the following extraction protocol. A
volume of 15 ml of 0.1 M phosphate buffer was added to each
microcosm, then the soil was resuspended by stirring for 2 min in a
vortex. Next, a volume of 10 ml of hexane was added to the soil
suspension in order to selectively extract the soil microorganisms
having a hydrophobic envelope. This mixture was stirred for 15 min
on a rotary stirrer. Each tube was centrifuged for 10 min at 4
000.times.g (swinging bucket rotor, Beckman GS-15R centrifuge) in
order to separate the various liquid phases and to sediment the
soil particles at the bottom of the tube. The interface containing
the targeted microorganisms is located between the aqueous phase
and the organic phase. It was removed with a pipette and a 1 ml
tip, the end of which was cut off, and then deposited on a 2.5 cm
Teflon filtering membrane with a porosity of 0.45 .mu.M (Durapore,
Millipore, France). The membrane was subjected to vacuum-suction
filtration in order to remove the liquid phase and to concentrate
the bacteria on the membrane. Said membrane was placed in a pill
bottle containing 1 ml of phosphate buffer for the decontamination
step.
[0122] Decontamination of the Extract and Counting
[0123] This step is very useful, or even essential, for removing
the interfering edaphic microbial flora from the soil. To do this,
the decontamination protocol combined an acid decontamination
(addition of 100 .mu.l of 4% hydrochloric acid, incubation for 20
min) with an estimated pH of 1.3 and a basic decontamination
(addition of 200 .mu.l of 4% sodium hydroxide, incubation for 20
min) with an estimated pH of 12.6, then the mixture was neutralized
by adding 100 .mu.l of hydrochloric acid before the addition of 1
ml of mixture of sodium hypochlorite and sodium hydroxide. After
incubation for 15 minutes, the supernatant was removed, deposited
on a new Teflon filtering membrane and rinsed with 20 ml of 0.1 M
phosphate buffer in order to remove the bleach. The membrane was
then placed in a new pill bottle containing 1 ml of enriched 7H9
and scraped with a loop (wire loop) in order to detach the cells
from the filter. For the purpose of counting the mycobacteria
extracted, 100 .mu.l of each final suspension were inoculated in
triplicate on enriched and supplemented 7H11, and incubated for two
months at 37.degree. C.
Example 2: Results
[0124] 2.1. Voltammetric Behavior of p-AP-OG and of p-AP-G
[0125] The voltammograms presented in FIG. 2 show that it is
possible to distinguish the voltammetric response of the p-AP-OG
substrate (S) from that of the p-AP-G product (P). Indeed, the
oxidation peaks detected at potentials of .about.+0.2 V and
.about.+0.4 V (vs. Ag/AgCl) were respectively recorded for p-AP-OG
and p-AP-G. This peak potential difference, linked to the presence
or absence of the octanoyl electron-withdrawing group, indicates
that p-AP-OG can be used as a substrate for the amperometric
detection of the acyltransferase activity of Ag85.
[0126] Indeed, as shown by the scheme in FIG. 1, Ag85 is capable of
hydrolyzing the ester bond of p-AP-OG and of transferring the
octanoyl group onto trehalose in order, respectively, to generate
p-AP-G and acyl trehalose according to a ping-pong mechanism. Thus,
the intensity of the p-AP-G oxidation peak measured at around
.about.+0.4 V vs. Ag/AgCl can be chosen as an analytic response
since its value is proportional to the amount of p-AP-G produced,
and thus to that of Ag85, and indirectly to that of the
mycobacteria present in the sample to be analyzed.
[0127] 2.2. Electrochemical Detection of the Acyltransferase
Activity of the Ag85 Protein
[0128] In order to demonstrate the acyltransferase activity of Ag85
in the presence of p-AP-OG and of an acyl group accepter, the
reaction mixture containing the Ag85 protein, p-AP-OG and trehalose
was incubated in the presence and absence of p-AP-G with stirring
for 4 hours at 37.degree. C. A negative control containing only
p-AP-OG was analyzed in parallel under the same conditions.
[0129] As shown by the series of voltammograms presented in FIG. 3,
the curve recorded for the negative control (curve as a line ---)
presents one p-AP-OG oxidation peak potential around .about.+0.2 V
vs. Ag/AgCl, whereas the voltammetric response obtained in the
presence of Ag85 (curve as a continuous line) possesses two
oxidation peaks located at around +.about.0.4 and 0.55 V vs.
Ag/AgCl, respectively.
[0130] A supplementary study (results not presented) showed that
the value of the p-AP-OG oxidation peak potential increases as a
function of the chloride ion concentration in the solution.
Moreover, the information regarding the commercially available Ag85
indicates that the protein was lyophilized from a buffer containing
0.1 M NaCl. Thus, the higher values of the oxidation peak
potentials of p-AP-OG and of p-AP-G recorded in the presence of
Ag85 are probably due to that of the chlorides in the solution.
[0131] In order to validate this hypothesis, Ag85 was also
incubated in the presence of trehalose and of a mixture of p-AP-OG
and p-AP-G. The comparison of the voltammograms (curve as a
continuous line and dotted curve) of FIG. 3 confirms the identity
of each peak shows that it is possible to envision electrochemical
detection of Ag85 using p-AP-OG as substrate.
[0132] 2.3. Electrochemical Detection of the Acyltransferase
Activity of Ag85 in a Liquid Culture of M. bovis BCG
[0133] For the purpose of applying the method for electrochemically
detecting Ag85 in order to demonstrate the growth of mycobacteria
in a liquid culture medium, the analysis of the supernatant of a
culture of M. bovis BCG strain Pasteur (avirulent model vaccine
strain of tuberculosis mycobacteria) and also that of the bacterial
pellet were envisioned. The concentrations of substrate and of
co-substrate, the enzymatic catalysis reaction time and also the
need to include a filtration step before the electrochemical
measurement were studied in a series of preliminary
experiments.
[0134] 2.3.1. Analysis of the Supernatant
1. It has been demonstrated that Ag85 is a major secretion product
of M. tuberculosis in the replicative phase (Wiker and Harboe,
Microbiol. Rev., 56(4): 648-661, 1992) [9]. The protocol
represented schematically in FIG. 4 and described in section 1.4
was carried out in order to analyze 5 ml of a culture of M. bovis
BCG containing 1.1.times.10.sup.7 cfuml.sup.-1. A negative control
(enriched 7H9 medium without bacteria) was analyzed in parallel
according to the same protocol and the results are presented in
FIG. 5. The voltammetric response of the negative control (curve as
a continuous line) corresponds overall to the p-AP-OG oxidation
peak located at around .about.+0.25 V vs. Ag/AgCl, whereas that
recorded for the culture supernatant (curve as a line ---) shows
not only the p-AP-OG oxidation peak (with a lower intensity), but
also the p-AP-G oxidation peak. The presence of the latter
indicates the presence of Ag85 in the culture supernatant and
therefore that of the mycobacteria in the culture analyzed.
[0135] 2.3.2. Analysis of the Bacterial Pellet
[0136] Since Ag85 is also involved in the repair and construction
of the wall of mycobacteria, the protocol represented schematically
in FIG. 6 was carried out in order to analyze 10 ml of M. bovis BCG
culture containing 2.times.10.sup.6 cfuml.sup.-1. In parallel, the
supernatant of this same culture was analyzed as previously.
[0137] The voltammogram (curve as a line ---) presented in FIG. 7
confirms the detection of the activity of Ag85 in the culture
supernatant with the presence of the p-AP-OG and p-AP-G oxidation
peaks. Furthermore, the voltammetric response recorded for the
analysis of the bacterial pellet (curve as a continuous line) shows
only the p-AP-G oxidation peak with a strong intensity. This result
indicates that all of the p-AP-OG substrate has been converted into
p-AP-G during the incubation step and thus suggests that the
bacterial pellet contains a larger amount of Ag85 than the
supernatant for a given culture volume. A supplementary study on
the specificity of the method showed that the acyltransferases
present in Pseudomonas aeruginosa, Pseudomonas fluorescens,
Pseudomonas monteilii, Staphylococcus aureus, Staphylococcus
epidermidis, Streptococcus pyogenes, Rhodococcus corallinus,
Achromobacter xylosoxidans, Citrobacter freundii, Enterobacter
cloacae, Enterococcus faecalis, Enterococcus faecium, Klebsiella
pneumoniae and Stenotrophomonas maltophilia are capable of
converting p-AP-OG into p-AP-G in the presence of trehalose.
[0138] Thus, the amperometric response of p-AP-G measured during
the analysis of the bacterial pellet of M. bovis BCG probably
results, on the one hand, from the acyltransferase activity of the
Ag85 presence in the mycobacterial envelope and, on the other hand,
from the acyltransferases contained in the mycobacteria.
[0139] 2.4. Electrochemical Detection of Several Species of
Mycobacteria in Liquid Cultures
[0140] The proof of concept of the electrochemical detection of
mycobacteria in the presence of p-AP-OG and trehalose was carried
out for the analysis of several species of mycobacteria that can be
handled in an L2 containment laboratory. The species selected are
those frequently found in respiratory specimens: M. intracellulare,
M. avium ssp. avium and M. xenopi. To do this, the protocol
described in section 1.4 was carried out and the results obtained
for the analysis of the bacterial pellets containing approximately
10.sup.6 bacilli are shown in FIG. 8. The obtaining of an oxidation
peak specific for the product of hydrolysis of the substrate by
Ag85 (p-AP-G) for the analysis of each species (curves as a line
---, E.about.+0.4 V vs. Ag/AgCl) confirms that it is possible to
detect, with an electrochemical method, the presence of
mycobacteria with p-AP-OG as substrate and trehalose as
co-substrate. For each species, quantification thresholds of
between 10.sup.3 and 10.sup.4 bacteriaml.sup.-1 of culture were
estimated.
[0141] 2.5. Detection of M. tuberculosis
[0142] The electrochemical detection of M. tuberculosis, a class 3
microorganism responsible for human tuberculosis, was carried out
by analyzing cultures of the H37Rv strain in 7H9 liquid medium and
on 7H11 agar medium according to the procedures of section 1.6. In
the case of liquid culture, the detection of Ag85 was envisioned in
the bacterial pellet and also in the culture supernatant.
[0143] With an oxidation peak specific for the p-AP-OG hydrolysis
product, the series of voltammograms presented in FIG. 9A confirms
that the M. tuberculosis strain produces Ag85 in a large amount in
the culture medium. These results confirm that it is possible to
envision the detection of M. tuberculosis via the electrochemical
measurement of the activity of Ag85 in the culture medium.
[0144] Moreover, the p-AP-G oxidation peaks recorded for the
analysis of the bacterial cells (FIGS. 9B, 9C; curves as a line
---) indicate that the electrochemical method involving p-AP-OG as
substrate and trehalose as acyl group accepter also makes it
possible to detect M. tuberculosis in the form of bacterial pellets
and of isolated colonies.
[0145] 2.6. Extraction and Detection of M. intracellulare in a
Respiratory Sample
[0146] In order to propose a method for direct (that is to say
without prior culture) electrochemical detection of mycobacteria in
samples of pulmonary origin (sputum, tracheal aspiration and
bronchial aspiration products), specimens from nontuberculous
patients were inoculated with a known amount of M. intracellulare
and analyzed according to the protocol of section 1.7.
[0147] Since the commercially available
fluidization-decontamination methods that are of use for the
preparation of the samples--which combine N-acetylcysteine and
sodium hydroxide--are not compatible with electrochemical detection
(poorly defined signals), the development of a protocol for
extracting the mycobacteria from specimens of pulmonary origin was
envisioned. The method proposed involves a hexane-isopropanol
mixture as extraction solvent. By precipitating the constituents of
the respiratory specimen, isopropanol makes it possible to get rid
of the viscous nature of the specimen, while the apolar solvent,
which hexane is, selectively extracts the mycobacteria, the wall of
which is very hydrophobic. Once recovered by filtration, the
mycobacteria were incubated with the substrate/co-substrate mixture
for the purpose of carrying out the electrochemical detection of
the acyltransferase activity (Ag85 and other enzymes present in the
mycobacterial cell).
[0148] The voltammograms presented in FIG. 10 show that it is
possible to detect the mycobacteria by virtue of the
electrochemical method in specimens of respiratory origin
previously treated with a hexane-isopropanol mixture. Indeed, the
oxidation peaks specific for the product of hydrolysis of p-AP-OG
by Ag85 is obtained for the sputum inoculated with M.
intracellulare (curve as a line ---, E.about.+0.4 V vs. Ag/AgCl)
while no oxidation peak linked to the presence of p-AP-G was
recorded for the non-inoculated sputum (curve as a continuous
line).
[0149] 2.7. Extraction and Detection of M. bovis BCG in a Soil
Sample
[0150] In order to evaluate the impact of the volume of hexane on
the extraction yield, sterile soil (5 g) was inoculated with the M.
bovis BCG strain (1.5.times.10 cfu per microcosm) and subjected to
the extraction protocol described in section 1.8 using hexane
volumes of 2 ml, 5 ml and 10 ml. The maximum extraction yield was
obtained using a hexane volume of 10 ml. Under these conditions,
50% of the mycobacteria that were inoculated in the microcosms were
extracted. This good extraction yield is linked to the high
affinity between hexane and the hydrophobic membrane of the
mycobacteria. Furthermore, hexane probably has a role in the
destruction of the bonds (physisorption, chemisorption) which bring
about adhesion of the mycobacteria with the soil particles.
[0151] The particular features of M. bovis, and in particular its
very slow growth, generally require a step of decontaminating the
extract before it is cultured, in order to remove the majority of
the endogenous microorganisms from the environmental substrates
while preserving the mycobacteria. Several protocols were evaluated
and only the one combining extreme pH variations and incubation in
the presence of sodium hypochloride was sufficiently effective for
destroying all the interfering microbial flora of the soil
co-extracted with the hexane at the same time as M. bovis BCG. By
applying the extraction-decontamination protocol described in
section 1.8 to sterile soil samples (5 g) inoculated with M. bovis
BCG (2.6.times.10.sup.6 cfu per microcosm), a yield of 2.5.+-.0.8%
(n=4) was obtained. This result suggests that the decontamination
is the limiting step in the approach since it removes 95% of the
mycobacteria that were extracted with hexane. This step is,
however, very useful, or even essential, for getting rid of the
contaminating microorganisms which have a hydrophobic envelope
similar to that of M. bovis, such as the various genera encountered
in Actinobacteria.
[0152] Although the method for extraction-decontamination of the
mycobacteria in soil of the invention makes it possible to recover
only approximately 2.5% of the bacteria inoculated into sterile
soil, this yield is much higher than that described in the
literature for the analysis of naturally contaminated environmental
samples with a magnetic immunocapture process (0.1%) (Sweeney et
al., Lett. Appl Microbiol., 43(4): 364-369, 2006; Sweeney et al.,
Appl Environ Microbiol., 73(22): 7471-7473, 2007) [10, 11].
[0153] 2.8. Quantitative Aspects of the Method
[0154] In order to take into account all of the random errors
during the implementation of the protocols, p-AP-OG was chosen as
an internal standard and the analytical response of the method was
defined as the ratio of the intensity of the p-AP-G oxidation peak
to the intensity of the p-AP-OG oxidation peak:
R = ip - AP - G ip - AP - OG ##EQU00001##
[0155] The parameters i.sub.p-AP-OG and i.sub.p-AP-G have been
defined on the voltammogram presented in FIG. 11.
[0156] As shown by the values of R collated in table 2 below, the
electrochemical method proposed by the inventors made it possible
to detect amounts of M. intracellulare of less than 100
bacilliml.sup.-1. This result makes it possible to envision
applications of the method for the analysis of actual samples,
after extraction of mycobacteria, without having recourse to a
prior culture step.
TABLE-US-00002 TABLE 2 Standardized analytical responses R (R.sub.0
corresponds to the analytical response registered for the culture
medium without bacteria) calculated from the voltammograms recorded
for the analysis of M. intracellulare at 10.sup.2, 10.sup.3,
10.sup.4, 10.sup.5 and 10.sup.6 bacilli ml.sup.-1 according to the
protocol described in section 1.4. M. intracellulare (bacillus
ml.sup.-1) R/R.sub.0 0 1 10.sup.2 2.9 10.sup.3 4.5 10.sup.4 7.4
10.sup.5 12.2 10.sup.6 40
[0157] Finally, a first repeatability study was carried out for the
analysis of the pellet of a culture of M. intracellulare containing
5.times.10.sup.6 bacilliml.sup.-1. A coefficient of variation of
94% was calculated for 5 repetitions.
[0158] 2.9. Application of the Method of Electrochemical Detection
of the Invention to the Monitoring of Mycobacterium tuberculosis
H37Rv Growth in Liquid Medium
[0159] 2.9.1. Comparison of the Time to Positivity of a Liquid
Culture of Mycobacterium tuberculosis H37Rv with the BD BACTEC.TM.
MGIT.TM. Automated Device
[0160] To do this, three volumes of medium (volumes, A, B and C)
were prepared for testing the detection method described in the
invention (Table 3).
TABLE-US-00003 TABLE 3 Volumes of the various reagents used to
prepare the culture media Electrochemical detection Reagents BACTEC
.TM. Volume A Volume B Volume C BD BACTEC .TM. 7 ml 3.5 ml 2 ml 1
ml MGIT .TM. medium Growth 0.8 ml 0.4 ml 230 .mu.l 115 .mu.l
supplement and BBL MGIT .TM. PANTA p-AP-OG 100 .mu.l 57 .mu.l 29
.mu.l 2 .times. 10.sup.-2M Trehalose 100 .mu.l 57 .mu.l 29 .mu.l 5
.times. 10.sup.-1M Final volume 7.8 ml 4.1 ml 2.344 ml 1.173 ml
[0161] An M. tuberculosis bacterial suspension was prepared then
diluted twice to one tenth in 7H9 culture medium (samples -1 and
-2). These diluted suspensions were used to inoculate the various
tubes.
[0162] The tubes incubated in the BACTEC.TM. automated device were
subjected to an automatic measurement of the fluorescence once an
hour. The time to positivity was expressed in days.
[0163] The tubes were analyzed daily by the method of the
invention, by taking a volume of 30 .mu.l of the culture and
depositing it at the surface of a screen-printed sensor without
prior treatment. The measurements were carried out by linear
voltammetry and the positivity of the sample corresponds to the
appearance of a p-AP-G oxidation peak at around .about.+0.50 V vs.
Ag/AgCl.
[0164] Since the electrochemical measurements were not carried out
continuously (once a day in the best of cases), there is for the
moment an uncertainty about the exact moment at which the
positivity appears, hence the expression of the results in the form
of .ltoreq.x days.
TABLE-US-00004 TABLE 4 Times to positivity obtained with the BACTEC
.TM. automated device and with the electrochemical method during
the incubation of control tubes and of tubes inoculated with M.
tuberculosis H37Rv (1 to 2 repetitions per sample) Electrochemical
detection (time in BACTEC .TM. days) Samples (time in days) Volume
A Volume B Volume C Control 1 Negative Negative Negative Negative
Control 2 Negative Negative -2 6.09 .ltoreq.7 .ltoreq.7 .ltoreq.4
-2 6.09 .ltoreq.7 .ltoreq.7 .ltoreq.3 -1 4.2 .ltoreq.4 .ltoreq.3
.ltoreq.2 -1 4.21 .ltoreq.4 .ltoreq.3 .ltoreq.2
[0165] The results of table 4 indicate that the electrochemical
method made it possible to monitor the culture of M. tuberculosis
in lower volumes (volume C) than the BACTEC.TM. method, and thus to
reduce the time to positivity of the sample (.ltoreq.2 days instead
of 4 days for the dilution -1 and .ltoreq.3-4 days instead of 6
days for the dilution -2).
[0166] 2.9.2. Comparison of the Time to Positivity of Four
Respiratory Samples Inoculated or not Inoculated with M.
tuberculosis H37Rv, after Fluidization-Decontamination with the BD
BACTEC.TM. MGIT.TM. Automated Device
[0167] Four respiratory samples were supplied by the CHU Dijon
(samples 1 and 2: fibroscopy, sample 3: bronchoalveolar lavage and
sample 4: sputum). For each sample, a 3 ml aliquot was contaminated
with 500 .mu.l of M. tuberculosis H37Rv suspension; a second
aliquot, which was not contaminated, served as a control. After a
fluidization-decontamination step (Biocentric NacPac kit), each
pellet was resuspended in 1.1 ml of culture medium. Two volumes of
500 .mu.l of the previous solution are then respectively introduced
into a BACTEC.TM. tube and a Falcon tube containing the medium A
(table 3).
[0168] The tubes incubated in the BACTEC.TM. automated device were
subjected to an automatic measurement of the fluorescence once an
hour. The time to positivity was expressed in days.
[0169] The cultures carried out in the volumes of medium A (table
3) were incubated at 37.degree. C. and analyzed with the
electrochemical method of the invention. The electrochemical
measurements and the interpretation thereof were carried out as in
section 2.9.1. above.
TABLE-US-00005 TABLE 5 Time to positivity of four respiratory
samples that were contaminated or not inoculated (control) in MGIT
tubes obtained with the electrochemical method and the BACTEC
automated device Electrochemical BACTEC .TM. detection Respiratory
(time in days) (time in days) samples Control Inoculated Control
Inoculated Sample 1 -- 9.15 -- .ltoreq.7 Sample 2 -- 4.16 --
.ltoreq.5 Sample 3 -- 5.07 -- .ltoreq.5 Sample 4 -- 5.20 --
.ltoreq.5
[0170] The results collated in table 5 show that the
electrochemical method was as effective as BACTEC.TM. for the
detection in liquid culture of respiratory samples artificially
contaminated with M. tuberculosis.
[0171] Finally, by combining the results of tables 4 and 5, the
electrochemical method of the invention is capable of demonstrating
more rapidly the growth of M. tuberculosis in a liquid culture
(.about.two times less time) and thus its presence in a respiratory
sample.
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* * * * *